Development of a facile genetic transformation system for the Spanish elite rice paella genotype Bomba.
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ABSTRACT:
SUBMITTER: Saba-Mayoral A
PROVIDER: S-EPMC9135871 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Transformation of commercially important indica cultivars remains challenging for the scientific community even though Agrobacterium-mediated transformation protocols for a few indica rice lines have been well established. We report successful transformation of a commercially important restorer line JK1044R of indica rice hybrid JKRH 401. While following existing protocol, we optimized several parameters for callusing, regeneration and genetic transformation of JK1044R. Calli generated from the rice scutellum tissue were used for transformation by Agrobacterium harboring pCAMBIA2201. A novel two tire selection scheme comprising of Geneticin (G418) and Paramomycin were deployed for selection of transgenic calli as well as regenerated plantlets that expressed neomycin phosphotransferase-II gene encoded by the vector. One specific combination of G418 (30 mg l(-1)) and Paramomycin (70 mg l(-1)) was very effective for calli selection. Transformed and selected calli were detected by monitoring the expression of the reporter gene uidA (GUS). Regenerated plantlets were confirmed through PCR analysis of nptII and gus genes specific primers as well as dot blot using gus gene specific as probe.
| S-EPMC4840146 | biostudies-literature
Project description:Key messageEstimating genetic gains and formulating a future salinity elite breeding panel for rice pave the way for developing better high-yielding salinity tolerant lines with enhanced genetic gains. Genetic gain is a crucial parameter to check the breeding program's success and help optimize future breeding strategies for enhanced genetic gains. To estimate the genetic gains in IRRI's salinity breeding program and identify the best genotypes based on high breeding values for grain yield (kg/ha), we analyzed the historical data from the trials conducted in the IRRI, Philippines and Bangladesh. A two-stage mixed-model approach accounting for experimental design factors and a relationship matrix was fitted to obtain the breeding values for grain yield and estimate genetic trends. A positive genetic trend of 0.1% per annum with a yield advantage of 1.52 kg/ha was observed in IRRI, Philippines. In Bangladesh, we observed a genetic gain of 0.31% per annum with a yield advantage of 14.02 kg/ha. In the released varieties, we observed a genetic gain of 0.12% per annum with a 2.2 kg/ha/year yield advantage in the IRRI, Philippines. For the Bangladesh dataset, a genetic gain of 0.14% per annum with a yield advantage of 5.9 kg/ha/year was observed in the released varieties. Based on breeding values for grain yield, a core set of the top 145 genotypes with higher breeding values of > 2400 kg/ha in the IRRI, Philippines, and > 3500 kg/ha in Bangladesh with a reliability of > 0.4 were selected to develop the elite breeding panel. Conclusively, a recurrent selection breeding strategy integrated with novel technologies like genomic selection and speed breeding is highly required to achieve higher genetic gains in IRRI's salinity breeding programs.
| S-EPMC10830834 | biostudies-literature
Project description:Four marine bacteria, Alteromonas sp. strains A27, A28, A29, and A30, that lyse the diatom Skeletonema costatum NIES-324 were isolated from coastal seawater samples. They were also able to lyse the diatoms Thalassiosira sp. and Eucampia zodiacs and the raphidophycean flagellate Chattonella antiqua. Cryptic indigenous plasmids, designated pAS28 and pAS29, were detected in Alteromonas sp. strains A28 and A29, respectively. These plasmids appeared to be similar based on size and restriction site analysis. A shuttle vector that replicates in Escherichia coli and Alteromonas sp. strain A28 was constructed by fusing pAS28 and E. coli vector pCRIIc. The 16-kbp chimeric plasmid, designated pASS1, had the ability to transform strain A28 at a frequency of 10(6) transformants per microg of DNA. Deletion analysis of pASS1 showed that the 4.7-kb EcoRI-HindIII region of pAS28 was essential for plasmid maintenance in strain A28. This EcoRI-HindIII fragment contained an open reading frame which appeared to encode a 708-amino-acid protein.
| S-EPMC106278 | biostudies-literature
Project description:Heterosis refers to the superior performance of hybrids relative to the parents. Utilization of heterosis has contributed tremendously to the increased productivity in many crops for decades. Although there have been a range of studies on various aspects of heterosis, the key to understanding the biological mechanisms of heterotic performance in crop hybrids is the genetic basis, much of which is still uncharacterized. In this study, we dissected the genetic composition of yield and yield component traits using data of replicated field trials of an "immortalized F(2)" population derived from an elite rice hybrid. On the basis of an ultrahigh-density SNP bin map constructed with population sequencing, we calculated single-locus and epistatic genetic effects in the whole genome and identified components pertaining to heterosis of the hybrid. The results showed that the relative contributions of the genetic components varied with traits. Overdominance/pseudo-overdominance is the most important contributor to heterosis of yield, number of grains per panicle, and grain weight. Dominance × dominance interaction is important for heterosis of tillers per plant and grain weight and has roles in yield and grain number. Single-locus dominance has relatively small contributions in all of the traits. The results suggest that cumulative effects of these components may adequately explain the genetic basis of heterosis in the hybrid.
| S-EPMC3465387 | biostudies-literature
Project description:Genetic transformation is crucial for both investigating gene functions and for engineering of crops to introduce new traits. Rice (Oryza sativa L.) is an important model in plant research, since it is the staple food for more than half of the world's population. As a result, numerous transformation methods have been developed for both indica and japonica rice. Since breeders continuously develop new rice varieties, transformation protocols have to be adapted for each new variety. Here we provide an optimized transformation protocol with detailed tips and tricks for a new African variety Komboka using immature embryos. In Komboka, we obtained an apparent transformation rate of up to 48% for GUS/GFP reporter gene constructs using this optimized protocol. This protocol is also applicable for use with other elite indica rice varieties.
| S-EPMC7842583 | biostudies-literature
Project description:most of the research concerning the influence of genetics on endurance performance has been carried out by investigating target genes separately. However, endurance performance is a complex trait that can stem from the interaction of several genes. The objective of this study was to compare the frequencies of polymorphisms in target genes involving cardiorespiratory functioning in elite endurance athletes vs. non-athlete controls. genotypic frequencies were determined in 123 elite endurance athletes and in 122 non-athletes. Genotyping of ACE (rs4340), NOS3 (rs2070744 and rs1799983), ADRA2a (rs1800544 and rs553668), ADRB2 (rs1042713 and rs1042714), and BDKRB2 (rs5810761) was performed by polymerase chain reaction. The total genotype score (TGS: from 0 to 100 arbitrary units; a.u.) was calculated from the genotype score in each polymorphism. the mean TGS in non-athletes (47.72 ± 11.29 a.u.) was similar to elite endurance athletes (46.54 ± 11.32 a.u., p = 0.415). The distribution of TGS frequencies were also similar in non-athletes and elite endurance athletes (p = 0.333). There was no TGS cut-off point to discriminate being elite endurance athletes. the genetic profile in the selected genes was similar in elite endurance athletes and in controls, suggesting that the combination of these genes does not determine endurance performance.
| S-EPMC8391315 | biostudies-literature
Project description:For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans, which employs a guide RNA to target an activator complex to the promoter region of a gene of interest, thus driving transcriptional expression of that gene. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust and constitutive overexpression. We further demonstrate the specificity of the system via RNA sequencing. We highlight the application of CRISPR activation to overexpress genes involved in pathogenesis and drug susceptibility, and contribute toward the identification of novel phenotypes. Consequently, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.
| S-EPMC9911074 | biostudies-literature
Project description:BackgroundThe plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize the infection process at the molecular biological level, no genetic transformation protocol has yet been established for F. avenaceum. In the current study, it is shown that F. avenaceum can be efficiently transformed by Agrobacterium tumefaciens mediated transformation. In addition, an efficient and versatile single step vector construction strategy relying on Uracil Specific Excision Reagent (USER) Fusion cloning, is developed.ResultsThe new vector construction system, termed USER-Brick, is based on a limited number of PCR amplified vector fragments (core USER-Bricks) which are combined with PCR generated fragments from the gene of interest. The system was found to have an assembly efficiency of 97% with up to six DNA fragments, based on the construction of 55 vectors targeting different polyketide synthase (PKS) and PKS associated transcription factor encoding genes in F. avenaceum. Subsequently, the ΔFaPKS3 vector was used for optimizing A. tumefaciens mediated transformation (ATMT) of F. avenaceum with respect to six variables. Acetosyringone concentration, co-culturing time, co-culturing temperature and fungal inoculum were found to significantly impact the transformation frequency. Following optimization, an average of 140 transformants per 106 macroconidia was obtained in experiments aimed at introducing targeted genome modifications. Targeted deletion of FaPKS6 (FA08709.2) in F. avenaceum showed that this gene is essential for biosynthesis of the polyketide/nonribosomal compound fusaristatin A.ConclusionThe new USER-Brick system is highly versatile by allowing for the reuse of a common set of building blocks to accommodate seven different types of genome modifications. New USER-Bricks with additional functionality can easily be added to the system by future users. The optimized protocol for ATMT of F. avenaceum represents the first reported targeted genome modification by double homologous recombination of this plant pathogen and will allow for future characterization of this fungus. Functional linkage of FaPKS6 to the production of the mycotoxin fusaristatin A serves as a first testimony to this.
| S-EPMC4133957 | biostudies-literature
Project description:BACKGROUND:A cytoplasm of CW-type cytoplasmic male sterile (CMS) line is derived from Oryza rufipogon strain W1 and fertility is restored by a single nuclear gene, Rf17. We have previously reported that CW-CMS were effective for breeding CMS lines of Indica Group rice cultivars, IR 24 and IR 64. The applicability of this CW-CMS/Rf17 system to produce other elite Indica Group rice cultivars with CMS was explored. FINDINGS:Out of seven elite Indica Group rice cultivars, complete CMS lines were obtained for six cultivars: NSIC Rc 160, NSIC Rc 240, Ciherang, BRRI dhan 29, NERICA-L-19, and Pusa Basmati. The fertility of these six lines was restored when Rf17 was present. A CMS line was not obtained for the cultivar Samba Mahsuri. CONCLUSIONS:The CW-CMS/Rf17 system will be useful to produce CMS lines and restorer lines of various elite Indica Group rice cultivars.
| S-EPMC6751230 | biostudies-literature
Project description:Here, we sought to assess target-specificity and detect potential off-target effects of a novel CRISPR activation (CRISPRa) system in Candida albicans. We used the CRISPRa system to target ALS1 for overexpression, and we also created a CRISPRa strain that does not target any gene for overexpression for use as a control. Differential gene expression analysis between the CRISPRa-ALS1 strain and the non-targeting strain allowed us to determine if ALS1 was the only gene overexpressed by our CRISPRa-ALS1 strain, or if the system non-specifically targeted other genes as well.
2022-05-03 | GSE201904 | GEO